According to the ribosome scanning model, traditional for most eukaryotic mRNAs, the 40S ribosomal subunit binds to the 5′-cap and moves along the nontranslated 5′-sequence until it reaches an AUG codon (Kozak (1986) Adv. Virus Res. 31: 229-292; Kozak (1989) J. Mol. Biol. 108: 229-241). Although for the majority of eukaryotic mRNAs only the first open reading frame (ORF) is translationally active, there are different mechanisms by which mRNA may function polycistronically (Kozak (1986) Adv. Virus Res. 31: 229-292). If the first AUG has unfavourable sequence context, 40S subunits may bypass it and initiate at downstream AUG codon (leaky scanning mechanism). Termination-reinitiation has also been suggested to explain the initiation of translation of functionally dicistronic eukaryotic mRNAs (Kozak (1989) J. Mol. Biol. 108: 229-241). Another mechanism for discontinuous ribosome migration (“shunting”) on mRNA has been recently proposed for cauliflower mosaic virus (CaMV) 35S RNA (Futerrer et al. (1993) Cell 73: 789-802).
In contrast to the majority of eukaryotic mRNAs, the initiation of translation of picornaviral RNAs takes place by an alternative mechanism of internal ribosome entry. A picornaviral 5′-nontranslated region (5′NTR) contains a so-called internal ribosome entry site (IRES) or ribosome landing pad (Pelletier and Sonenberg (1988) Nature 334: 320-325; Molla et al. (1992) Nature 356: 255-257) which is folded into a complex secondary structure and contains a pyrimidine-rich tract followed by an AUG codon (Agol (1991) Adv. Virus Res. 40: 103-180; Wimmer et al. (1993) Annu. Rev. Genet.27: 353-436; Sonennberg and Pelletier (1989) BioEssays 11: 128-132). Internal ribosome entry has also been reported for other viral (Le et al. (1994) Virology 198: 405-411; Gramstat et al. (1994) Nucleic Acid Res. 22: 3911-3917) and cellular (Oh et al. (1992) Gen Dev. 6: 1643-1653) RNAs.
It is important to emphasize that the picornaviral and other known IRESes are not active in plant cell systems.
The genome of tobamoviruses (TMV UI is the type member of a group)) contains four large ORFs. In vitro translational experiments have shown that the two components of the replicase (the 130K and its read-through 183K proteins) are translated directly from the genomic RNA (Pelham and Jackson (1976) Eur. J. Biochem 67: 247-256). The other two proteins (30K movement protein, MP, and coat protein, CP) are translated from two individual subgenomic RNAs (sgRNAs). The structurally dicistronic I2 sgRNA is translated to give the 30 K MP, while its 3′-terminal CP gene is silent and a monocistronic sgRNA codes the CP (Palukaitis and Zaitlin (1986) in The Plant Viruses, eds. Van Regenmortel and M. Fraenkel-Conrat, 2: 105-131, Plenum Press).
Recently a new tobarnovirus, crTMV, has been isolated from Olearacia officinalis L. plants and the crTMV genome has been sequenced (6312 nucleotides) (Dorokhov et al. (1993) Doklady of Russian Academy of Sciences 332: 518-522; Dorokhov et al. (1994) FEBS Lett. 350: 5-8). A peculiar feature of crTMV is its ability to infect systemically the members of Cruciferae family. The crTMV RNA contains four ORFs encoding the proteins of 122 K (ORF1), 178 K (ORF2), the readthrough product of 122 K, 30 K MP. (ORF3) and 17 K CP (ORF4). Unlike other tobamoviruses, the coding regions of the MP and CP genes of crTMV overlap for 25 codons, i.e. 5′ of the CP coding region are sequences encoding MP.
We have reported recently that translation of the 3′-proximal CP gene of crTMV RNA occurs in vitro and in planta by a mechanism of internal ribosome entry which is mediated by a specific sequence element, IRESCP (Ivanov et al. (1997) Virology 232: 32-43).
Our results indicated that the 148-nt region upstream of the CP gene of crTMV RNA contained IRESCP,148CR promoting internal initiation of translation in vitro. Dicistronic IRESCP,148CR containing chimeric mRNAs with the 5′-terminal stem-loop structure preventing translation of the first gene, expressed the CP or β-glucuronidase (GUS) genes despite their 3′-proximal localization. The capacity of crTMV IRESCP for mediating internal translation in vitro distinguishes this tobamovirus from the well known type member of the genus, the TMV UI. However, in the present invention we show that the 148-nt sequence upstream from CP gene of TMV UI is capable of expressing moderately the 3′-proximal gene from dicistronic construct in transformed yeast cells, i.e. this sequence can be termed IRESCPUI. We found that the 75-228-nt region upstream of the MP gene of crTMV, TMV UI and cucumber green mottle mosaic virus contains IRESes that allow 5′-end-independent internal initiation of translation on dicistronic mRNAs containing IRES as the intercistronic spacer.
The present invention shows that genomes of tobamoviruses contain the IRES-elements upstream of both genes: the MP and CP genes capable of promoting the 3′-proximal gene expression from bicistronic mRNAs. Therefore, this invention relates to a novel functional activity of nucleotide sequences located upstream of the MP and/or CP genes of tobamoviruses: their ability to mediate the cap-independent expression of the 5′-distal genes being inserted as an intercistronic spacers in bi- (or polycistronic) eukaryotic mRNAs.
The tobamoviruses provide new examples of internal ribosome entry sites which are markedly distinct from IRESes shown for picomaviruses and other viral and eukaryotic mRNAs. The tobamovirus IRESes described in this invention are the first IRES sequences functional in plant cells described so far. In addition, the tobamovirus genome-derived IRES elements were shown to be functional in animal and yeast cells.